1. Field of the Invention
The present invention relates to a solution based method for carbon-centered radical mediated protium/heavy hydrogen exchange into the reduced carbon atoms in a molecule of interest. The methods of the present invention can be used to determine which reduced carbon atoms in a molecule are solvent accessible. In particular, the methods of the present invention can be used to determine which carbon atoms in a macromolecule, such as a peptide or protein, are solvent accessible when the macromolecule is in a particular folded state.
2. Background Art
Carbon-centered radical mediated heavy hydrogen labeling of compounds is well known in the art. For example, radiolysis studies have demonstrated that hydroxyl (OH) radical can act as a hydrogen atom abstractor that removes a hydrogen atom from reduced carbon atoms in molecules such as amino acids, peptides, and proteins to form a carbon-centered radical. See e.g. Garrison, 1987, Chem. Rev. 87:381-398; von Sonntag, 1987, The Chemical Basis of Radiation Biology, Taylor and Francis: London. However, determining which carbon atoms in a molecule react with the hydroxyl radical has been elusive. In the case of DNA, reaction of DNA with a hydrogen atom abstractor results in strand scission. Thus, the site of the reaction between a hydrogen atom abstractor, such as hydroxyl radical, and the DNA can be inferred by studying DNA cleavage patterns. Hertzberg et al., Biochemistry 23:3934-3945.
The predominant mode of hydrogen atom abstractor initiated damage of DNA and proteins is removal of a hydrogen atom from a Cxe2x80x94H bond of a reduced carbon atom to produce the corresponding carbon-centered radical. von Sonntag, supra. The carbon-centered radical has various chemical fates including: (1) reaction with molecular oxygen, initially forming a hydroperoxyl species that can result in hydroxylation (Fu et al., 1995, Biochem. J. 311:821-827) and DNA (Breen et al., 1995, Free Radic. Biol. Med. 18:1033-1077) or protein (Davies, J. Biol. Chem. 262: 9895-9901) strand scission, (2) recombination of two carbon-centered radicals to form a carbon-carbon crosslink (Karam et al., 1984, Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 46:715-724; Davies et al., 1987, J. Biol. Chem. 262:9902-9907; Gajewski et al., Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 54:445-449); and (3) chemical repair by H atom donation, such as that mediated by sulfhydryls (Alexander et al., 1955, Radiobiology Symposium, pp 49-55, Bacq and Alexander, Academic Press: New York). Thus, the initial site of hydroxyl radical attack on a molecule is often obscured by the multiplicity of resulting products.
In DNA, the abstraction of any ribose hydrogen atom and subsequent oxidation leads to chain scission. Thus, carbon-centered radical mediated assays provide a general method for identifying residues that react with the hydrogen atom abstractor. The ability to randomly initiate cleavage of the DNA backbone by enzymatic or chemical means is the most essential chemical step in DNA footprinting. The sites protected by protein binding are excluded from solvent and consequently, are not susceptible to attack by the hydrogen atom abstractor. The absence of these DNA product fragments in electrophoretic separations identify the DNA nucleotides involved in protein-DNA recognition. The footprint resolution is dependent upon the chemical nature of the DNA cleaving reagent. Base resolution can be achieved by using a small, sterically unhindered molecule that is highly reactive and is nonspecific, indiscriminately cleaving at all base positions of the DNA backbone. Hydroxyl radical generated by xcex3-radiolysis has been experimentally shown to possess all of these attributes and has been used to identify contacts in protein-DNA complexes at base resolution. Franchet-Beuzig, et al., 1993, Biochemistry 32:2104-2110.
Despite the success in using carbon-centered radical mediated reactions in DNA footprinting techniques, analogous footprinting techniques to study protein-protein interactions have proven to be unsatisfactory. Small metal chelates have been used to randomly cleave polypeptide chains. The chelate, iron(II)-EDTA, has been utilized in either a tethered or untethered form. The tethered form can be used to map its proximity to neighboring peptide bonds. Rana et al., 1990, J. Am. Chem. 112:2457-2458. Using untethered iron(II)-EDTA as a nonspecific protein cleaving agent, Heyduk et al. have studied solvent-accessible sites induced by changes in protein conformation upon ligand binding for cAMP receptor protein in the presence and absence of cAMP. Heyduk et al., 1994, Biochemistry 33:9643-9550. In addition, Greiner and coworkers have used iron(II)-EDTA as a nonspecific protein cleaving agent to map the interactions between the subunits of E. coli RNA polymerase. Greiner et al, 1996, Proc. Natl. Acad. Sci., U.S.A. 93:71-75. In both cases, the peptide fragments were electrophoretically separated and visualized by immunostaining with antibodies specific to the N- and/or C-terminal peptides of the protein. The limitations of this method are (1) iron(II)-EDTA cleavage tends to occur at hypersensitive sites, (2) antibodies for the N- and/or C-termini are required for the proteins of interest, and (3) identification of the sites of protection is usually confined to segments of 10-15 residues in length. Although this protein footprinting methodology permits mapping contact regions of protein domains involved in macromolecular assemblies, the ability of the technique to specifically identify the sites involved in recognition at the amino acid residue level has not been satisfactory.
The failure to achieve single residue resolution in protein footprinting studies despite the success in analogous DNA footprinting studies can also be understood by comparing the reactivity of hydrogen atom abstractors, such as hydroxyl radical, with proteins and DNA. For duplex DNA, hydroxyl radical react with the macromolecule by abstracting a hydrogen atom from solvent-accessible Cxe2x80x94H bonds of the deoxyribose ring along the DNA backbone, producing a carbon-centered radical that reacts with O2 and results in strand scission. Breen et al., 1995, Free Radic. Biol. Med. 18:1033-1077. Cleavage of globular proteins occurs by a similar mechanism. Stadtman, 1993, Annu. Rev. Biochem. 62:797-821. Abstraction of a Cxcex1xe2x80x94H of the protein backbone by hydroxyl radical produces a carbon-centered radical that reacts with O2, forming a hydroperoxyl species that leads to protein strand scission. However, the majority of solvent-accessible Cxe2x80x94H bonds present on the protein""s solvent-accessible surface are not comprised of the backbone (Cxcex1xe2x80x94Hxcex1) but those of the side chains. Thus, in protein footprinting the major pathway of hydrogen abstractor reactivity with proteins is not exploited.
The reaction of hydroxyl radical with alkyl Cxe2x80x94H bonds is rapid, 108 Mxe2x88x921sxe2x88x921 (Buxton et al., 1988, (J. Phys. Chem. Ref. Data 17:513-886) a value 10-100 fold less than the diffusion limit. This indicates that a hydrogen atom abstraction occurs on average every 10-100 collisions. This high frequency of reaction prevents hydroxyl radical generated in bulk solution from diffusing into the interior of macromolecular complexes. The success of DNA footprinting with hydroxyl radical demonstrates that formation of macromolecular complexes protects the residues at the molecular interface from reacting with hydroxyl radical. Tullius et al, 1987, Methods of Enzymology 155:537-558, Wu Ed., Academic Press: New York.
Electron spin resonance studies have also used carbon-centered radical mediated labeling of compounds to study molecules of interest, such as macromolecules. In EPR studies, a hydrogen atom abstractor, such as hydroxyl radical, is used to generate the carbon-centered radical of the molecule of interest. This highly unstable carbon-centered radical is then reacted with a spin trapping agent such as a nitrone. Buettner et al, 1990, Methods in Enzymology 186:127-133, Packer and Glazer, Eds., Academic Press: New York. Spin trapping agents are bulky and typically have a mass that exceeds 100 Daltons. The reaction of the carbon-centered radical with the spin trapping agent results in the covalent attachment of the spin trapping agent to the molecule of interest. In the case of molecules of interest such as peptides and proteins, the covalent attachment of bulky spin trapping agents is particularly unsatisfactory because it tends to reduce the solubility of the macromolecule, induces the macromolecule to adopt a nonphysiological conformation, and disrupts potential interactions, such as protein/protein or protein/drug interactions, that are the subject of the investigation.
Rather than using bulky spin trapping agents as taught by EPR studies, it is desirable to use a heavy hydrogen donor to xe2x80x9crepairxe2x80x9d carbon-centered radicals by donating a heavy hydrogen to the carbon-centered radical as shown FIG. 1. The advantage of such a repair reaction is that the molecule of interest is labeled with heavy hydrogen rather than a bulky spin trapping agent. Thus, the conformation of the molecule of interest is not altered and physiologically relevant information may be obtained. A second major advantage is that because this reaction is an isotope exchange reaction, the chemical nature of the molecule of interest is unchanged. This permits multiple solvent accessible reduced carbon atoms to be monitored in a single molecule, thus enhancing the sensitivity of the method over that of EPR studies. While such a xe2x80x9crepairxe2x80x9d approach is appealing in theory, reduction of such a reaction to practice has been particularly problematic, especially for molecules of interest that have low solubility in solution. One obstacle to achieving satisfactory results is that the hydrogen atom abstractor used to generate a carbon-centered radical in the molecule of interest tends to preferentially react with the heavy hydrogen donor rather then the molecule of interest. Another obstacle is that exposure of the molecule of interest to hydrogen atom abstractors such as hydroxyl radical tends to decompose the molecule of interest.
Goshe et al., June 1997, describes research directed to addressing the specific obstacles that prevent the attainment of satisfactory results from carbon-centered radical mediated hydrogen/heavy hydrogen labeling of molecules of interest. Goshe et al., June 1997, Meeting Abstract, American Society of Mass Spectroscopy, 45th Annual Conference. Goshe et al., June 1997, used hydrogen atom abstractors, such as radiolysis generated hydroxyl radical, to remove hydrogens from reduced carbon atoms in free amino acids. This work raised the possibility that hydroxyl radical may be capable of abstracting a hydrogen atom from a Cxe2x80x94H bond of the amino acids leucine and valine, producing a carbon-centered radical that is quenched by a heavy hydrogen donor via heavy hydrogen donation to the C-centered radical by a heavy hydrogen donor. However, even in the simple system described by Goshe et al., June 1997, conventional heavy hydrogen donors, such as ascorbic acid, do not provide for a satisfactory amount of heavy hydrogen incorporation into the molecule of interest. Goshe et al., June 1997, found that using dithiothreitol as a heavy hydrogen donor, rather than ascorbic acid, resulted in improved heavy hydrogen incorporation levels in molecules of interest such as the side chains of the free amino acids leucine and valine. Although Goshe et al., June 1997, supra, teaches an improved heavy hydrogen donor reagent, the conditions taught by Goshe et al., June 1997, remain unsatisfactory for the general study of molecules of interest. Under the conditions of Goshe et al., June 1997, the hydrogen atom abstractor preferentially oxidizes the heavy hydrogen donor rather than the molecule of interest. As a result, the heavy hydrogen donor supply in the reaction is rapidly depleted and the highly unstable carbon-centered radicals are not repaired with a hydrogen isotope. Rather, the carbon-centered radicals undergo a variety of undesirable reactions such as hydroxylation and crosslinking which degrades and/or denatures the molecule of interest. Further, if the hydrogen atom abstractor is hydroxyl radical concomitantly generated by radiolysis, solvated electrons generated by the radiolysis tend to also degrade the molecule of interest. This is particularly true of proteins and peptides, which the solvated electron readily reacts with, resulting in reductive cleavage of the amide bonds in the backbone. In simple systems, where the molecule of interest is highly soluble in solution and readily available, the problems provided by the Goshe et al., June 1997, reaction conditions can be partially offset by raising the concentration of the molecule of interest in the solution to a very high level. Because a high concentration of the molecule of interest is present in the solution, the small amount of sample that survives the reaction may be sufficient to detect heavy hydrogen incorporation. However, the partial remedy of increasing the concentration of the molecule of interest in the reaction is not a general solution to the problems presented by the Goshe et al., June 1997, reaction conditions because more complex molecules, such as peptides and proteins, do not have the solubility or stability required to overcome the problems presented by the Goshe et al., June 1997, reaction conditions.
According to the above background, there is a need for an improved method for the carbon-centered radical-mediated heavy hydrogen labeling of reduced carbon atoms in molecules of interest.
This invention provides an improved method for the carbon-centered radical mediated heavy hydrogen labeling of reduced carbon atoms in molecules of interest. Using the methods of the present invention, molecules of interest, including complex macromolecules such as peptides and proteins, can by studied using carbon-centered radical mediated heavy hydrogen labeling techniques. The methods of the present invention can be used to determine which reduced carbons of a molecule of interest are solvent accessible. Further, the methods of the present invention may be used to characterize amino acid residues that are involved in peptide-protein, protein-protein, and/or protein-drug interactions. The methods of the present invention have general utility in the field of life sciences. In particular, the methods of the present invention have significant utility in the fields of biochemistry, structural biology, and rational drug design.
According to the methods of the present invention, a solution containing a heavy hydrogen donor, a heavy hydrogen source, and a molecule of interest is prepared. Dissolved oxygen is removed from this solution, typically by bubbling the solution with an oxygen-free gas. When a substantial amount of oxygen has been removed from the solution, a hydrogen atom abstractor, such as hydroxyl radical, may then be generated in the solution by various methods disclosed herein. The hydrogen atom abstractor removes hydrogen atom from solvent accessible reduced carbons presented by the molecule of interest. As depicted in FIG. 1, the removal of hydrogen atoms from the molecule of interest results in the formation of the corresponding carbon-centered radical. The heavy hydrogen donor present in the solution repairs the carbon-centered radical using available sources of hydrogen present in the solution, including the heavy hydrogen source. Thus, heavy hydrogen is incorporated into a high percentage of the carbon-centered radicals. Because the reaction of the hydrogen atom abstractor with reduced carbons such as those found in alkyls is rapid, the method of the present invention is particularly effective at selectively labeling solvent accessible reduced carbons atoms. An additional feature of the present invention is that the labeling reaction is fast. The rate of the labeling reaction is limited by the rate of reaction of the heavy hydrogen donor with the carbon-centered radical. Thus if a heavy hydrogen donor having a very fast rate constant, such as dithiothreitol, is chosen the labeling reaction may be completed within milliseconds. Once the carbon atoms in the molecule of interest have been labeled with heavy hydrogen, using the methods of the present invention, the location of the heavy hydrogen can be determined by a variety of methods including electrospray ionization-mass spectroscopy, scintillation counting and/or NMR methods.
In a preferred embodiment, the hydrogen atom abstractor is generated using radiolysis. Radiolysis is a preferred technique for generating hydroxyl radical because the rate at which hydroxyl radical is generated in solution by various radiation sources has been accurately determined. Another major advantage of radiolysis is that it requires no additional chemical other than the water necessarily present in aqueous solution. Thus, it is possible to use a radiation source, such as 137Cs xcex3-ray source to generate hydroxyl radical in a solution at a very precise rate. By integrating this rate of hydroxyl radical generation over time, the total equivalent concentration of hydroxyl radical generated in a solution can be precisely and accurately determined. This has the advantage of making carbon-centered radical mediated heavy hydrogen labeling experiments highly reproducible. In addition, for a given molecule of interest, a series of labeling experiments using varying total equivalent concentrations of hydroxyl radical can be performed in order to provide an additional dimension of information about the solvent accessibility of particular solvent accessible carbon atoms in a molecule of interest.
If radiolysis is used to generate the hydrogen abstractor, in the methods of the present invention, the solution should be provided with an electron scavenger source prior to exposing the solution to the radiation source. The electron scavenger source absorbs the damaging free electrons that are generated in the solution by the radiation source. If N2O gas is used to remove a substantial amount of oxygen from the solution, then the N2O that dissolves into the solution as the N2O gas is bubbled into the solution serves as a preferred electron scavenger source.
In a preferred embodiment, the solution includes an internal reference. The internal reference serves to normalize the effective hydroxyl radical dose between successive labeling experiments. The internal reference is a molecule having reduced carbon atoms that readily exchanges with heavy hydrogen using the methods of the present invention. A preferred internal reference is leucine or norleucine.
In another preferred embodiment, the carbon-centered radical mediated heavy hydrogen labeling reaction is repeated a multiple number of times in succession on the same sample. Between each exchange reaction, additional reduced heavy hydrogen donor is added to the sample solution to compensate for the heavy hydrogen donor in the sample that is lost during the labeling reaction. Additionally, if the hydrogen atom abstractor is hydroxyl radical generated by radiolysis, additional amounts of electron scavenger source is added to the solution between exchange reactions to compensate for depletion of the electron scavenger source in the exchange reaction. In a preferred embodiment, this electron scavenger source is provided by bubbling the solution with N2O gas.
In yet another preferred embodiment, the molecule of interest is a peptide or protein. The exchange reaction isotopically labels particular solvent accessible side chains on the peptide or protein. The determination of the amino acid residues containing the isotopic label provides a means of assigning residues of proteins as solvent accessible and can be employed to study protein conformational changes and protein-protein interactions at the amino acid level. The formation of stable carbon-heavy hydrogen bond using carbon-hydrogen/heavy hydrogen exchange has the advantage over amide hydrogen/heavy hydrogen exchange of (1) producing highly stable carbon-heavy hydrogen label and (2) selectively targeting the heavy hydrogen label to solvent accessible side chains, rather than just amide backbones.